Biology subjects

Tsunoyama, T. A.

Publications and source records attributed to Tsunoyama, T. A..

7 recordsLinked to original sources

bioRxiv2025

SynGAP forms biocondensates at sub-micromolar concentrations and recruits PSD95 and receptor oligomers, functioning as a key initiator of PSD formation

A key issue in neuronal circuit regulation is how synapse formation is initiated. Synapse formation could start when one or more synaptic scaffold proteins that can initiate synapse formation reach certain threshold concentrations in the dendritic shaft, which might lead to their oligomerization or even liquid-liquid phase separation (LLPS). By combining in vitro reconstitution of purified proteins with live-cell single-molecule and confocal imaging, we demonstrated that SynGAP alone forms assemblies of nanoscale clusters containing several to several tens of molecules at 10-nM order concentrations and micron-scale LLPS hydrogel-like condensates at submicromolar concentrations. The trimers of SynGAP's intrinsically disordered region (IDR) induced by its coiled-coil domain are responsible for SynGAP condensation. CaMKII-mediated phosphorylation moderately suppresses SynGAP condensation, and also increases condensate liquidity. While PSD95 fails to form assemblies under these conditions, it is recruited to SynGAP condensates by specifically binding to the PDZ-binding motif of SynGAP. SynGAP[PSD95] condensates selectively immobilize postsynaptic transmembrane proteins, Neuroligin1 and AMPAR-TARP2 complexes, in a manner dependent on their oligomerization state, indicating cooperative recruitment dynamics among SynGAP, PSD95, and transmembrane components, which might mimic initial PSD assembly. These findings suggest that SynGAP may act as a primary nucleator of postsynaptic density assembly, challenging the PSD95-centered models.

neuroscience↗

bioRxiv2024

Single-molecule detection of transient dimerization of opioid receptors 1: Homodimers' effect on signaling and internalization

Opioid receptors (ORs) are critical for endogenous and synthetic analgesics. Their homodimerization is considered important for their pharmacological diversities, but whether they form homodimers remains controversial. Here, we established that the three classical ORs, -, {kappa}-, and {delta}-ORs (MOR, KOR, and DOR, respectively) undergo repeated transient (120-180 ms) homodimerizations every few seconds. This was done by using single-molecule imaging and developing theories for analyzing single-molecule colocalization data, which provide the key parameters, homodimer-monomer dissociation equilibrium constants and rate constants. Their 9-26 amino-acid C-terminal cytoplasmic domains, without sequence similarities, are involved in specific homodimerization, whereas the transmembrane domains provide less specific affinities. Using the membrane-permeable peptides mimicking the C-terminal homodimerization sequences which block homodimerizations, functions of monomers and homodimers were dissected. KOR and DOR homodimers, but not MOR homodimers, activate downstream G-proteins differently from monomers upon agonist addition, without influencing OR internalization. These findings could guide strategies to enhance OR-based analgesia.

biophysics↗

bioRxiv2024

Single-molecule detection of transient dimerization of opioid receptors 2: Heterodimer blockage reduces morphine tolerance

Heterodimerization of opioid receptors (ORs), MOR, KOR, and DOR, is implied in their functional regulation and diversification, and thus its understanding is crucial for developing better analgesic treatments. However, our knowledge on OR heterodimerization/heterodimers remains limited. Here, using single-molecule imaging and functional analysis, we found that MOR, the main morphine receptor, repeatedly forms transient ({approx}250 ms) heterodimers with DOR every 1-10 seconds, but not with KOR, whereas DOR and KOR also form transient heterodimers. We obtained all the heterodimer-monomer equilibrium constants and rate constants with/without agonists. We identified the critical heterodimer binding sites in the extracellular domains, in addition to the less-specific transmembrane domains, and developed soluble peptide blockers for MOR-DOR and DOR-KOR heterodimerization, using amino-acid sequences mimicking the extracellular binding sites. With these peptide blockers, we dissected the monomer/dimer roles in OR internalization and signaling. The soluble MOR-DOR heterodimer blocker reduced the development of long-term morphine tolerance in mice.

biophysics↗

bioRxiv2021

iTRVZ: liquid nano-platform for signal integration on the plasma membrane

Cell survival in immune-competent organisms relies on both growth and immune evasion, yet these processes have been studied as independent cellular programs. Here, using super-resolution single-molecule imaging, we identify GEM (Growth and Evasion Metastable hub), a {approx}34-nm, metastable, nano-liquid, plasma membrane hub that physically integrates receptor-type tyrosine kinase (RTK) inputs with CD59-mediated immune-evasion signaling within the same assembly. GEM is formed by LLPS-like protein clustering on PI(4,5)P2-containing raft-nanodomains. Upon concurrent RTK and CD59 stimulations, GEM recruits both receptors and downstream kinases, enabling reciprocal activation via nano-liquid confinement that supralinearly amplifies PLC{gamma}-IP3-Ca2+ and PI3K-Akt survival signaling outputs. In mice, disrupting GEM suppresses tumor growth in vivo, establishing nano-liquid GEM as an integrator that physically couples growth and CD59-mediated immune-evasion signaling for cell survival.

cell biology↗

bioRxiv2021

Development of ultrafast camera-based imaging of single fluorescent molecules and live-cell PALM

The spatial resolution of fluorescence microscopy has recently been greatly improved. However, its temporal resolution has not been improved much, despite its importance for examining living cells. Here, by developing an ultrafast camera system, we achieved the worlds highest time resolutions for single fluorescent-molecule imaging of 33 (100) {micro}s (multiple single molecules simultaneously) with a single-molecule localization precision of 34 (20) nm for Cy3 (best dye found), and for PALM data acquisition of a view-field of 640x640 pixels at 1 kHz with a single-molecule localization precision of 29 nm for mEos3.2. Both are considered the ultimate rates with available probes. This camera system (1) successfully detected fast hop diffusion of membrane molecules in the plasma membrane, detectable previously only by using less preferable 40-nm gold probes and bright-field microscopy, and (2) enabled PALM imaging of the entire live cell, while revealing meso-scale dynamics and structures, caveolae and paxillin islands in the focal adhesion, proving its usefulness for cell biology research. SummaryAn ultrafast camera developed by Fujiwara et al. allows single fluorescent-molecule imaging every 33 s with a localization precision of 34 nm (every 100 s; 20 nm), and enables ultrafast PALM imaging of whole live cells.

cell biology↗

bioRxiv2021

Focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion

Using the ultrafast camera system and new theories for hop diffusion described in the companion paper, we for the first time demonstrated that membrane molecules undergo hop diffusion among the compartments in the bulk basal plasma membrane (PM), with virtually the same compartment sizes (108 nm) as those in the bulk apical PM and the same dwell lifetimes within a compartment (10 and 24 ms for the phospholipid and transferrin receptor, respectively), suggesting that the basic structures and molecular dynamics are very similar in the bulk regions of the apical and basal PMs. Ultrafast PALM and single-molecule imaging revealed that the focal adhesion (FA) is mostly a fluid membrane, partitioned into [~]74-nm compartments where transferrin receptor and {beta}3 integrin undergo hop diffusion, and that the FA membrane is sparsely dotted with 51-nm diameter paxillin islands, where many other FA proteins probably assemble (compartmentalized archipelago model). {beta}3 integrin intermittently associates with the paxillin islands, dynamically linking them to the extracellular matrix. SummaryAn ultrafast camera with single fluorescent-molecule sensitivities developed by Fujiwara et al. reveals that the focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion, and integrin {beta}3 molecules become temporarily immobilized at the islands.

cell biology↗

bioRxiv2020

High-speed single-molecule imaging reveals signal transduction by induced transbilayer raft phases

Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59-cluster rafts and GM1-cluster rafts stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK, continually recruited Lyn and H-Ras right beneath them in the inner leaflet, with dwell lifetimes of <0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the non-raftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Since GM1-cluster rafts recruited Lyn and H-Ras as efficiently as CD59-cluster rafts, and the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipid-anchored signaling molecules by lateral raft-lipid interactions, and thus serve as a key signal transduction platform. SummaryHigh-speed single-molecule imaging indicated that CD59-cluster rafts and GM1-cluster rafts stably induced in the plasma membrane outer leaflet generated nano-scale transbilayer raft phases, which continually and transiently recruited Lyn and H-Ras in the inner leaflet by cooperative raft-lipid interactions.

cell biology↗